The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer

Abstract

The recent cataloging of the genomic aberrations in breast cancer has revealed the diversity and complexity of the disease at the genetic level. To unravel the functional consequences of specific repertoires of mutations and copy number changes on signaling pathways in breast cancer, it is crucial to develop model systems that truly recapitulate the disease. Here we discuss the three-dimensional culture models currently being used or recently developed for the study of normal mammary epithelial cells and breast cancer, including primary tumors and dormancy. We discuss the insights gained from these models in regards to cell signaling and potential therapeutic strategies, and the challenges that need to be met for the generation of heterotypic breast cancer model systems that are amenable for high-throughput approaches.

Keywords: Breast cancer; Co-culture; Microenvironment; Signaling pathway; Three-dimensional cell culture; Tissue architecture.

Fig. 1. De novo synthesis of extracellular matrix components of cultured normal mammary epithelial cells is dependent on the substratum

(A) Normal mammary epithelial cells cultured on tissue culture plastic secrete laminin and other ECM proteins such as fibronectin and type IV collagen but fail to organize, whereas (B) normal mammary epithelial cells grown cultured on type I collagen gels deposit an endogenously synthesized basement membrane and recapitulate their in vivo phenotype [31,32].

Fig. 2. Three-dimensional cell culture systems

For the study of breast cancer, (A) Conventional monolayer cell cultures [120,121,146]; (B) Spheroid cultures, where cells spontaneously aggregate either growing in low attachment plates (left) or hanging drops (right) [80,118,138,147]; (C) 3D lrECM cultures, where cells are either embedded (left) or growing on top of the ECM overlaid with a dilute solution of lrECM (right) [39,40,49,111]; and (D) Natural or synthetic polymers and macroporous scaffolds support the formation of 3D structures of cells [52,97], have been employed.

Fig. 3. Stiffness of tissues and culture model systems

Stiffness (or elastic modulus), measured in Pascals (Pa), of normal mouse mammary gland, mouse tumors, and matrices and plastic used in cell culture models. Data from [55,128]. BM, basement membrane.

Fig. 4. Complex three-dimensional cell culture model: microvascular niche

Lung-like microvascular niches were created by co-culture of lung fibroblasts and human umbilical vein endothelial cells (see [81]). This assay was used to demonstrate that the microvascular endothelium (denoted by CD31 immunostaining, red) steers malignant, genotypically aberrant breast epithelial cells (T4-2 cells, green, as indicated by white arrowhead) into a quiescent state characterized by a lack of Ki-67 immunostaining (magenta). In this assay, a subset of lung fibroblasts express alpha-smooth muscle actin (cyan) and associate intimately with the microvasulature, behaving as pericytes. Scale bar=100 μM. (C.M. Ghajar & M.J. Bissell, unpublished data [81]).

Fig. 5. Screen to identify genes conferring resistance to signaling inhibitors

Breast cancer cells, such as T4-2 cells, form unpolarized tumor-like structures when grown in 3D lrECM cell cultures. Treatment with signaling inhibitors, such as small molecule EGFR tyrosine kinase inhibitors, induces reversion of the tumor-like structures to a polarized acinar phenotype (right). A disorganized unpolarized structure of breast cancer cells transduced with a cDNA library is selected after signaling inhibitor treatment, and the cDNA insert sequenced to identify genes conferring resistance to reversion and signaling inhibition (left) [110].